Biochemistry (Moscow) (v.74, #11)

Inhibition of C5-cytosine-DNA-methyltransferases by O. V. Kirsanova; N. A. Cherepanova; E. S. Gromova (1175-1186).
Changes in the methylation pattern of genomic DNA, particularly hypermethylation of tumor suppressor genes, occur at early stages of tumor development. Errors in DNA methylation contribute to both initiation and progression of various cancers. This stimulates significant interest in searching for inhibitors of C5-DNA-methyltransferases (MTases). Here we review the known nucleoside mechanism-based reversible and irreversible inhibitors of the MTases, as well as non-nucleoside ones, and discuss their inhibitory mechanisms and application for MTase investigations and cancer therapy.
Keywords: DNA methyltransferase; methylation pattern; carcinogenesis; gene inactivation; inhibitors of DNA-methyltransferases; mechanism of inhibition; cancer therapy

Production of biologically active human myelocytokines in plants by A. S. Zvereva; L. E. Petrovskaya; A. V. Rodina; O. Y. Frolova; P. A. Ivanov; L. N. Shingarova; T. V. Komarova; Y. L. Dorokhov; D. A. Dolgikh; M. P. Kirpichnikov; J. G. Atabekov (1187-1194).
An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.
Keywords: granulocyte colony-stimulating factor; granulocyte macrophage colony-stimulating factor; viral vector; agroinjection

Use of models of biomacromolecule separation in AMT database generation for shotgun proteomics by M. L. Pridatchenko; I. A. Tarasova; V. Guryca; A. S. Kononikhin; C. Adams; D. A. Tolmachev; A. Yu. Agapov; V. V. Evreinov; I. A. Popov; E. N. Nikolaev; R. A. Zubarev; A. V. Gorshkov; C. D. Masselon; M. V. Gorshkov (1195-1202).
Generation of a complex proteome database requires use of powerful analytical methods capable of following rapid changes in the proteome due to changing physiological and pathological states of the organism under study. One of the promising technologies with this regard is the use of so-called Accurate Mass and Time (AMT) tag peptide databases. Generation of an AMT database for a complex proteome requires combined efforts by many research groups and laboratories, but the chromatography data resulting from these efforts are tied to the particular experimental conditions and, in general, are not transferable from one platform to another. In this work, we consider an approach to solve this problem that is based on the generation of a universal scale for the chromatography data using a multiple-point normalization method. The method follows from the concept of linear correlation between chromatography data obtained over a wide range of separation parameters. The method is further tested for tryptic peptide mixtures with experimental data collected from mutual studies by different independent research groups using different separation protocols and mass spectrometry data processing tools.
Keywords: high performance liquid chromatography; proteomics; mass spectrometry

Primary processes of charge separation in reaction centers of YM210L/FM197Y and YM210L mutants of Rhodobacter sphaeroides by A. G. Yakovlev; L. G. Vasilieva; A. Y. Shkuropatov; V. A. Shuvalov (1203-1210).
Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll B A at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* → P+B A and to the absence of stabilization of separated charges in the state P+B A . Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule PB of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm−1 in the single mutant YM210L to ∼100 cm−1 in the double mutant. Oscillations with 100–150 cm−1 frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P+B A state of both mutants reflect a motion of the PB molecule relatively to PA in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the B A absorption band are conserved within a shorter time ∼0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.
Keywords: photosynthesis; charge separation; reaction center; wave packet; electron transfer

Myoglobin and mitochondria: Oxymyoglobin interacts with mitochondrial membrane during deoxygenation by G. B. Postnikova; S. V. Tselikova; E. A. Shekhovtsova (1211-1218).
The rates of oxygen uptake by rat liver mitochondria (MC) (native coupled, freshly frozen, and uncoupled by FCCP) have been measured polarographically in the absence (V 0) or presence (V 1) of 0.11–0.25 mM sperm whale MbO2. Under the same standard conditions, the rate of sperm whale MbO2 deoxygenation (V 2) has been studied spectrophotometrically in the presence of respiring MC. For freshly frozen MC, the dependence of V 1 and V 2 on the overall charge of MbO2 has been investigated at pH 5.6–7.6, and the influence of other differently charged proteins (apomyoglobin, egg lysozyme, lactalbumin, and BSA) has been studied at pH 7.4. It is shown that the rate of mitochondrial respiration in the presence of MbO2 increases by 10–30% (V 1 > V 0). No myoglobin effect is observed for FCCP-uncoupled MC (V max does not change). The rate of MbO2 deoxygenation is equal to the rate of oxygen uptake by mitochondria (V 2/V 1 ∼ 1 at pH 7.2–7.5). At varying pH < 7.2, the V 2 values become markedly higher than V 1, evidently due to the increased MbO2 positive charge and its stronger interaction with negatively charged mitochondrial membrane. At pH 7.4, on the contrary, V 2 is twice lower than V 1 in the case of negatively charged CM-MbO2 (pI 5.2), which has carboxymethylated histidines. Positively charged lysozyme (pI 11) strongly inhibits MbO2 deoxygenation (V 2) without affecting oxygen uptake by MC (V 0 and V 1). At the same time, apomyoglobin (pI 8.5), which is structurally very similar to the holoprotein, and both negatively charged lactalbumin (pI 4.4) and BSA (pI 4.7) have no substantial influence on V 2 and V 1. The MC membrane evidently has no specific sites for the interaction with myoglobin. Rather, the protein contacts with phospholipids of the outer membrane during MbO2 deoxygenation, and electrostatic interactions are of great importance for this process.
Keywords: mitochondria; myoglobin; deoxygenation; membrane

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.
Keywords: thioflavin T; amyloid; yeast cell wall protein; Bgl2p; Saccharomyces cerevisiae

In this work, the diameters of protein complexes formed upon interaction of ceruloplasmin (CP) with lactoferrin (LF) and myeloperoxidase (MPO) were determined. Gage dependence of the diameter of protein particles (myoglobin, albumin, LF, CP, MPO, aldolase, ferritin) on their molecular mass logarithm was calculated. The diameter of a complex formed upon mixing CP and LF was 8.4 nm, which is in line with the radius of gyration obtained previously when the 1CP-1LF complex was studied by small-angle X-ray scattering. The diameter of a complex formed upon interaction of CP with MPO is 9.8 nm, corresponding to the stoichiometry 2CP: 1MPO. The diameter of a complex formed when LF is added to the 2CP-1MPO complex is 10.7 nm. The latter is consistent with the notion of a pentameric structure 2LF-2CP-1MPO with molecular mass of about 585 kDa.
Keywords: ceruloplasmin; lactoferrin; myeloperoxidase; protein-protein interaction; photon correlation spectroscopy

The differentiation status of fibroblasts can be characterized by their ability to induce Ah-receptor-dependent genes. The ability to induce Ah-receptor-dependent genes encoding cytochrome P450 isoforms, Ah-receptor repressor, and NADPH-quinine oxidoreductase were studied in the transformed cell clone K8 obtained from immortalized embryonic rat fibroblasts by treatment with benzo(a)pyrene and in the parental clone F27. Treatment with benz(a)anthracene did not induce the genes in the transformed clone K8 on passages 4–14, but the induction was recorded in the transformed clone beginning from the 16th passage and later, whereas in F27 cells the induction was observed throughout the experiment. Induction levels of mRNA of the induction-regulating genes encoding the Ah-receptor and Ah receptor nuclear translocator were similar in F27 cells and in the transformed cell clone K8 in both early and late passages. Electrophoretic mobility shift assay showed that in clone K8 transmission of the induction signal was disturbed in the early passages before interaction of the activated Ah-receptor with the recognizing region of DNA. Possible mechanisms responsible for the absence of induction in the early passages in the transformed cells are discussed.
Keywords: cell transformation; cytochrome P450; Ah-receptor; enzyme induction

Halogenated pyruvate derivatives as substrates of transketolase from Saccharomyces cerevisiae by O. A. Esakova; L. E. Meshalkina; G. A. Kochetov; R. Golbik (1234-1238).
Pyruvate derivatives halogenated at C3 were shown to be donor substrates in the transketolase reaction. No drastic differences between the derivatives were observed in the value of the catalytic constant, whereas the Michaelis constant increased in the following order: Br-pyruvate < Cl-pyruvate < Cl2-pyruvate < F-pyruvate < Br2-pyruvate. The presence of the halogenated pyruvate derivatives increased the affinity of apotransketolase for the coenzyme; of note, the extent of this effect was equal with both of the active centers of the enzyme. In contrast, the presence of any other substrate known to date, including hydroxypyruvate (i.e. pyruvate hydroxylated at C3), induced nonequivalence of the active centers in that they differed in the extent to which the affinity for the coenzyme increased. Consequently, the β-hydroxyl of dihydroxyethylthiamine diphosphate (an intermediate of the transketolase reaction) played an important role in the phenomenon of non-equivalence of the active centers associated with the coenzyme binding. The fundamental possibility was demonstrated of using halogenated pyruvate derivatives as donors of the halogen-hydroxyethyl group in organic synthesis of halogenated carbohydrates involving transketolase.
Keywords: transketolase; halogenated pyruvate derivatives; organic synthesis; CD spectra; nonequivalence of active centers

Proteomic analysis of human skeletal muscle (m. vastus lateralis) proteins: Identification of 89 gene expression products by M. A. Kovalyova; L. I. Kovalyov; I. Yu. Toropygin; S. V. Shigeev; A. V. Ivanov; S. S. Shishkin (1239-1252).
Proteins from bioptates and autoptates of human skeletal muscle m. vastus lateralis were separated by O’Farrell two-dimensional gel electrophoresis (2DE). MALDI-TOF MS and MS/MS enabled identification of 89 protein spots as expression products of 55 genes. A modification of the O’Farrell’s method including non-equilibrium electrophoresis in a pH gradient allowed detection — among major sarcomeric, mitochondrial, and cytosolic proteins — of several proteins, such as PDZ- and LIM domain-containing ones (pI > 8.70), fragments of known proteins, and a stable complex of heavy and light ferritin chains. The data underlie further studies of human skeletal muscle proteins in terms of molecular mechanisms of some physiological and pathological processes.
Keywords: proteins of human skeletal muscle; proteomics

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family by I. R. Grin; P. G. Konorovsky; G. A. Nevinsky; D. O. Zharkov (1253-1259).
Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K m = 150 nM and k cat = 1.2 min−1, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al3+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+, and Fe2+ in Tris-HCl buffer and by Cd2+, Zn2+, Cu2+, and Fe2+ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I50 for NEIL1 inhibition were 7 µM for Cd2+, 16 µM for Zn2+, and 400 µM for Cu2+. The inhibition of NEIL1 by Cd2+, Zn2+, and Cu2+ was at least partly due to the formation of metal-DNA complexes. In the case of Cd2+ and Cu2+, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.
Keywords: oxidative stress; DNA repair; DNA glycosylases; heavy metals

Sorbitol content was determined in the digestive gland of freshwater snail (Viviparus viviparus L.) in different seasons and in a short-term experiment on the water temperature decrease and on intoxication with cadmium chloride. In the model experiments, changes in activities of enzymes involved in sorbitol metabolism (acid phosphatases, sorbitol dehydrogenase, and aldose reductase) were also studied. Sorbitol was accumulated by the snail in response to the temperature decrease (as a cryoprotectant) and under conditions of acute intoxication (as a probable metabolic regulator or a nonspecific protective factor). However, the mechanisms of this accumulation are different: on cold adaptation sorbitol is produced as a result of reduction of glucose under the influence of aldose reductase, and on intoxication sorbitol is mainly produced from fructose under the influence of sorbitol dehydrogenase. Pathways of the sorbitol accumulation and its re-involvement into metabolism are not always the same, and this might be a mechanism for regulation of carbohydrate metabolism (at the initial stage of glycolysis) on adaptation to unfavorable factors of the environment.
Keywords: mollusks; cold adaptation; intoxication; cryoprotectants; sorbitol metabolism

To improve its stability and lipophilicity, Cu,Zn-superoxide dismutase (SOD) was chemically modified with linoleic and α-linolenic acids using two different methods. Higher retained enzymatic activity has been observed compared with SOD modified by macromolecular substances. Enhanced heat stability, acid and alkali resistance, and anti-pepsin/trypsin ability of the modified SOD were observed compared with those of the natural enzyme, the apparent oil-water partition coefficient being especially increased. The results characterize SOD modified with polyunsaturated fatty acids as a promising pharmacological tool.
Keywords: Cu,Zn-superoxide dismutase; polyunsaturated fatty acids; chemical modification; stability; partition coefficient

Identification of messenger RNA substrates for mouse T-STAR by L. Y. Zhang; M. Zeng; P. Chen; H. Q. Sun; D. C. Tao; Y. Q. Liu; L. Lin; Y. Yang; S. Z. Zhang; Y. X. Ma (1270-1277).
Using the method of isolation of specific nucleic acids associated with proteins (SNAAP), we have identified 10 candidate target mRNA substrates bound by mT-STAR (mouse T-STAR protein) from testis extract. Among them, our study focused on Fabp9, a gene that is essential for male gametogenesis, and showed that mT-STAR could directly bind to Fabp9 mRNAs. The binding sites are in a short sequence of the coding region and 3′ untranslated region of Fabp9 mRNA. These suggest that mT-STAR can regulate the metabolism and expression of Fabp9. In conclusion, identification of mT-STAR-bound mRNA substrates might help to illustrate the potential spectrum of the process and provide valuable insight into the biological function of this RNA-binding protein in spermatogenesis.
Keywords: Fabp9 ; mRNA substrates; mT-STAR; SNAAP; spermatogenesis

Interaction of DNA topoisomerase 1 with DNA intermediates and proteins of base excision repair by N. A. Lebedeva; N. I. Rechkunova; K. Agama; Y. Pommier; O. I. Lavrik (1278-1284).
The interaction of human recombinant DNA topoisomerase 1 (Top1) with linear and circular DNA structures containing a nick or short gap but lacking a specific Top1 recognition site was studied. The effect of key excision repair proteins on formation of the Top1 covalent adduct with the DNA repair intermediates was shown. Partial inhibition of the Top1-DNA-adduct formation upon addition of poly(ADP-ribose) polymerase 1 in the absence of NAD+ was shown, whereas in the presence of NAD+ formation of a high molecular weight product, most likely corresponding to poly(ADP)-ribosylated Top1-DNA adduct, was observed. The data show that the key base excision repair proteins can influence formation of suicide Top1-DNA adducts. Top1 was identified by immunoprecipitation in the bovine testis nuclear extract as the protein forming the main modification product with nick-containing DNA.
Keywords: DNA topoisomerase 1; DNA repair; base excision repair factors

Isotachophoresis of nucleic acids in agarose gel rods by V. N. Kondratova; I. V. Botezatu; V. P. Shelepov; A. V. Lichtenstein (1285-1288).
A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.
Keywords: DNA isotachophoresis; circulating DNA; agarose gel; DNA isolation; PCR

Glycoscience. Chemistry and chemical biology by G. Wiederschain (1289-1289).